Bioterror exposures include the possibility of exposure to either individual or combinations of biologic and radiation agents. After a documented or suspected Bioterror exposure, there is a variable period of time during which it is not clear what agent was used or what therapy would be most specific. In this situation it may not be known what specific therapy may be most effective, such as an antiviral or antibiotic. During this period and regardless of what specific agent is chosen, antioxidant support in the form described in this invention may be useful to support the immune system and improve the likelihood of survival of the affected individual.
The biology with the bacteria Bacillus anthracis and smallpox Smallpox virus, variola are reviewed as background for the invention.
In the early stage of exposure to anthrax the bacteria is usually in the spore form. The body uses the macrophage to phagocytize the spore and it is carried to regional lymph nodes. The formation of vegetative organisms thus occurs in the lymphatic system and spreads to the bloodstream. In a small number of cases, systemic anthrax can lead to meningeal involvement by means of lymphatic or hematogenous spread. At autopsy a classic finding of anthrax infection is the finding of extensive hemorrhage of the leptomeninges, which gives them a dark red appearance described as “cardinal's cap.”
In cases of pulmonary anthrax, peribronchial hemorrhagic lymphadenitis blocks pulmonary lymphatic drainage, leading to pulmonary edema. Death results from septicemia, toxemia, or pulmonary complications and can occur one to seven days after exposure.
Illness and death related to infection with the bacteria Bacillus anthracis is mediated by the interaction of three toxins formed by anthrax. The first is an antiphagocytic capsule (poly-n-glutamic acid), edema toxin and lethal toxin. The antiphagocytic factor inhibits phagocytosis and locks onto the scavenging cell called macrophage. This is followed by the insertion of the two other toxins, lethal factor toxin and edema toxin. The lethal factor appears to mediate the rapid onset of shock and death associated with systemic anthrax. While edema toxin causes swelling in all cells, lethal toxin exerts its effect selectively on the macrophage, even though the lethal toxin is injected into all cells (Hanna P et al, “On the Role of Macrophages in Anthrax,” Proc Natl Acad Sci (PNAS) USA: Vol. 90; 10198-10201, 1993).
The presence of low levels of toxin in macrophages stimulates the production of cell signaling hormones called cytokines such as IL-1 and TNF. At low levels these cytokines stimulate the immune response of the host, but at higher levels they mediate damage to the host, even extending to fatal shock (Hanna P et al, “On the Role of Macrophages in Anthrax,” PNAS USA: Vol. 90; 10198-10201, 1993). It is postulated that the macrophage accumulates the IL-1 until a critical level of toxin is reached which causes lysis of the cell and uncontrolled release of the accumulated IL-1 in a large amount that can lead to shock and death. The accumulation of IL-1 in many macrophages coupled with the contemporaneous release from many cells can result in death being the presenting symptom of anthrax infection (Hanna Pet al, PNAS USA: Vol. 90; 10198-10201, 1993).
Thus the macrophage plays a contradictory role. With most infections the macrophage plays a protective role ingesting and incapacitating invading organisms. With anthrax the normal protective mechanism has been mal-appropriated to facilitate the lethality of the anthrax toxin. The macrophage becomes the mediator of the lethality related to the toxin.
Macrophage disruption is mediated by an increase in the formation of Reactive Oxygen Species also known as Free Radicals. At high levels the toxins cause destruction of the macrophages. At lower levels the toxin-mediated free radicals stimulates these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. Antioxidants have been shown to moderately inhibit anthrax toxin-induced cytokine production in vitro. NAC also blocked the production of TNF-alpha in rat peritoneal macrophages activated with endotoxin (Pahan K, “N-acetyl cysteine inhibits induction of NO production by endotoxin or cytokine stimulated rat peritoneal macrophages C6 glial cells and astrocytes,” Free Radic Biol Med, 24(1):39-48 1998 Jan. 1). Mice pretreated with N-acetyl-L-cysteine showed partial protection against anthrax toxin exposure (Hanna P et al, “Role of macrophage oxidative burst in the action of anthrax lethal toxin,” Molecular Medicine (Mol Med) 1994 November; 1 (1):7-18).
Increasing the level of cysteine, a precursor of glutathione reduces the production of the cytokines and TNF-α that caused the body more harm than benefit. The increase in glutathione precursor also increases the potential for surviving toxin exposure in animals (Hanna P et al, “Role of macrophage oxidative burst in the action of anthrax lethal toxin,” Mol Med 1994 November; 1(1):7-18). Other studies have also demonstrated that reactive oxygen species are involved in the deterioration of cardiac function seen in endotoxin shock and their effect can be diminished by the use of antioxidants (Pattanaik U et al, “Reactive oxygen species and endotoxic shock: effect of dimethylthiourea,” J Cardiovasc Pharmacol Ther 2001 July; 6(3):273-85). The use of NAC prior to exposure to anthrax or its toxins have already been demonstrated to help reduce the negative effects of ROS and the release of cytokines in cell studies. NAC prior to exposure reduces the lethality of anthrax in animal studies.
While Hanna et al suggest positive effect in mice by NAC on exposure to toxins and on survivability of macrophages in culture,” (Hanna P et al, “Role of macrophage oxidative burst in the action of anthrax lethal toxin,” Mol Med 1994 November; 1(1):7-18), no reference cites a method or combination of NAC with antibiotics to treat anthrax in humans or mammals. Dixon, Meselson, Guillemin and Hanna, “Anthrax,” N. Engl. J. Med. 341(11): 815-26, Sep. 9, 1999 specifically note “Unfortunately, anti-toxin preparations are not currently available in the United States. Based on the findings of the animal and cell studies, glutathione and glutathione precursors containing cysteine or its physiologic form cystine represent a potent strategy for ameliorating the effects of inhalation anthrax.
Viruses are strands of information, either RNA or DNA, that depend on living cells to provide either the additional components or an environment which allow the virus to reproduce. Since they neither breathe nor reproduce on their own they are not considered living. After using the host cell machinery and enzymes to make new copies of itself, viruses program the production of a new covering which allow it to be released out of the cell and accepted by the next cell. To accomplish replication most viruses need to use the information contained in the DNA of the cell. Smallpox viruses are unusual in that they contain all of the information that they need to replicate, however it still needs the interior of the cell to replicate. Scientists who study the evolution of viruses conjecture that the information in the smallpox virus may have belonged to another organism that has now become incorporated in the DNA replication machinery of our cells (Takemura M, “Poxviruses and the origin of the eukaryotic nucleus,” J Mol Evol. Vol. 52(5):419-25, May 2001.). Viruses cause problems by changing the machinery of the cell that it has commandeered and by creating so many copies of itself that the cell cannot maintain its normal function, ceases functioning normally and may even rupture, releasing large amounts of virus.
Smallpox virus, called variola, is a DNA virus containing all the information that is necessary to reproduce; however it must be incorporated into a cell for reproduction to occur. This allows for the virus to replicate in the cytoplasm of a cell and to utilize the cell wall machinery to form a capsule and be released rapidly. The virus, after replicating in various tissue cells and creating a large number of copies, is released into the bloodstream before invading the skin and, in most cases, developing the skin lesions, or pocks that give rise to the common name for the disease state triggered by the virus: smallpox. While this is happening, the patient may not feel ill. Usually 12 days after exposure a feverish illness appears. Two to three days later the characteristic skin rash may appear. The rash appears as small pink spots called macules and progresses to enlarged, slightly raised papules. The papules progress to blisters. Eventually the blisters become turbid, looking like pustules, which were previously called pocks and are the characteristic feature for which the virus is named. The pustules then dry up and shrink, leaving a hard crust that eventually flakes off leaving a sunken scar. The distribution of the rash is characteristic of smallpox with the head and extremities affected more than the trunk.
Usually the severity of the disease follows the severity of the rash, but severe illness may precede the rash with the patient becoming prostrate before the outbreak of a rash. It is also possible to spread the virus without developing the rash, which means that outbreaks can be precipitated by individuals without rash or even severe illness.
Mortality: There is no treatment recognized once the illness has started. There are reports of mortality of 30% or more in unvaccinated populations (Henderson D A et al, “Consensus statement regarding Smallpox,” J. Am. Med. Assoc. Vol. 281(22):2127-37, Jun. 9, 1999).
Poxviruses have caused infections in man documented as far back as the Egyptian pharaohs. The Pharaoh Ramses V was apparently inflicted with the virus at the time of his death in 1157 B.C. Smallpox inflicted Europe in 710 A.D. Cortez carried smallpox to the America's in 1520 and precipitated the death of 2 million Aztecs in the following 2 years. In the cities of 18th century Europe, smallpox reached plague proportions as the highly infectious disease affected rich and poor alike, with five reigning monarchs dieing with the disease (http://www.tulane.edu/˜dmsander/WWW/335/Poxviruses.html). The last outbreak of small pox occurred in modern times in Somalia in 1977.
While most pox diseases, such as chickenpox, occur in man as well as animals, only smallpox uses humans as its reservoir. The selectivity of smallpox has allowed the success of an aggressive immunization campaign by the World Health Organization to eradicate the disease. The structure of the poxviruses are similar, which allows the use of a similar, but less virulent virus to effect a cross reacting immunization. In addition, smallpox causes only acute disease and does not set up dormant disease such as the Herpes virus does. The result is that individuals who survive a smallpox infection have immunity that lasts for life.
The fact that the animal poxviruses share similar biochemical characteristics on their covers that are recognized by the immune system allowed Edward Jenner to demonstrate the concept of vaccination in 1796 (Id). It had been observed that the milkmaids, while frequently affected with cowpox, rarely if ever became infected with smallpox. Jenner used cowpox, obtained from a milkmaid to inoculate an 8-year-old boy. Later, Jenner challenged the boy with an inoculation of smallpox and demonstrated that the immunization led to protection from the infection.
Vaccination became common around the world beginning around 1800, but smallpox was not eradicated until the World Health Organization made a major commitment to eradicate the virus with a worldwide vaccination campaign. Vaccination is done with a less virulent virus called vaccinia. The vaccinia virus used in modern vaccinations may have developed through the serial passage from arm to arm since the time of Jenner.
Smallpox causes serious infections with skin lesions and it is spread easily through respiratory contact with cough droplets. As a biological weapon, smallpox represents a serious threat as it has been reported to carry a case-fatality rate of 30% or more in unvaccinated populations. In addition there is no antiviral therapy specific for smallpox (Henderson D A et al, “Consensus statement regarding Smallpox,” Jour Amer Med Assoc. Vol. 281(22):2127-37, Jun. 9, 1999). The ability of an unvaccinated individual thus becomes dependent on the capacity of the individuals immune system to respond to the virus.
While smallpox is unique in its ability to replicate in the cytoplasm of a cell, it has common features with other viruses that make it identifiable by the immune system. Apparently the viral covering is different from self-tissue to the extent that an immune response is created to the viral cover. This is the foundation for the use of the vaccinia virus as a vaccine to stimulate antibodies to smallpox. It is the ability of the immune system to process the viral information, produce antibodies and coordinate the immune cell response for removal of virus that makes the immune system an effective protector against viruses.
Enhancing immune cell function with nutrient materials such as N-Acetyl-cysteine, which increase glutathione in the body and inside cells, will increase the immune surveillance and response mechanism and increase the likelihood of surviving a smallpox infection. Further, even for weakened virus, or cross-reacting immunization, N-Acetyl cysteine, which increases glutathione in the body and inside cells, increases and favorably influences immune response and lessens the likelihood of complications from vaccination.
Exposure of cells to virus decreases the intracellular glutathione, starting immediately after virus challenge. In a study with the herpes simplex virus type 1 (HSV-1) the addition of glutathione to the culture was not only able to restore the intracellular glutathione levels to near normal levels, but was also able to inhibit over 99% of the replication of HSV-1 (Palamara A T et al., “Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication,” Antiviral Res, Vol. 27(3):237-53 1995). Additionally, it was observed that the inhibition was concentration dependent and was maintained even if the glutathione was added as late as 24 hours after virus challenge, when the virus was fully established. Studies of the HSV-1 infected cells showed that glutathione dramatically reduced the number of extracellular and intracytoplasmic virus particles. Nucleocapsids were still detected within the nuclei of glutathione treated cells. The decrease in virus replication was related to a decrease in glycoprotein B, which is crucial for the release and infectivity of the virus. This suggests that glutathione inhibits the replication of HSV-1 by interfering with very late stages of the virus life cycle and not by interfering with cellular metabolism Id.
Viral pathogenesis and mutation is increased by free radical mediated oxidation (Akaike T, “Role of free radicals in viral pathogenesis and mutation Review Medical Virology,” Vol. 11(487-101, March-April 2001). The mechanism may be related to the depletion of antioxidant with resulting increase in oxidation. The increase in oxidation decreases the available glutathione. The oxidation stress status of the individual has been demonstrated to affect the pathogenicity of virus in animal studies (Beck M A et al, “Host nutritional status and its effect on a viral pathogen,” J Infect Disease Vol. 182 Suppl 1:S93-96, September 2000).
It is well documented that cellular immune function, termed T helper cell type 1 (TH1) function, is affected by the level of glutathione. Antigen presenting cells deficient in glutathione predispose to the formation of a TH2 response with a decrease in TH1 or cell mediated immune response (Peterson J D et al, “Glutathione levels in antigen-presenting cells modulate TH1 versus TH2 response pattern,” Proc Natl Acad Sci USA, Vol. 95(6):3071-6, Mar. 17, 1998). Cytotoxic T cell function, which is important in the management of viral infection, is decreased by 30% in animals deficient in glutathione (Lawrence B P et al, “Gamma-glutamyltranspeptidase knockout mice as a model for understanding the consequences of diminished glutathione on T cell-dependent immune responses,” European Journal Immunology Vol. 30(7):1902-10, July 2000). Thus, maintaining the glutathione level will help maintain a more favorable balance in the immune responses and an increase in survival. NAC has been demonstrated to increase and maintain the intracellular level of glutathione and protect against intracellular oxidative damage (Afaq F et al, “N-acetyl L-cysteine attenuates oxidant-mediated toxicity induced by chrysotile fibers,” Toxicology Letters Vol. 117(1-2):53-60, Sep. 30, 2000).
Intravenous NAC will improve survival of individuals infected with the smallpox virus by either of two methods.                1. NAC may contribute to a direct reduction in viral replication        2. NAC improves the immune surveillance and responsiveness of the immune system.        
In addition, regardless of exposure, whether it is bacterial or viral, NAC has been shown to be of benefit in the reducing the ventilator time in individuals with sepsis who require respiratory support. Intravenous NAC has been shown in humans with adult respiratory distress syndrome in patients with septic shock to be effective in reducing length of stay in the intensive care unit from 32 days to 13 days (Spapen H et al, “Does N-acetyl-L-cysteine influence cytokine response during early human septic shock?,” Chest, Vol. 113(6):1616-24 June, 1998. One of the beneficial mechanisms of NAC demonstrated in the study was the reduction in the production of the interleukin IL-8, a potential mediator of lung injury. Intravenous NAC infusion to lab rats after exposure to endotoxin reduced the features associated with septic shock (Schmidt W et al, Intensive Care Med, Vol. 24(9):967-72 Sep. 1998.
Studies have also demonstrated that reactive oxygen species are involved in the deterioration of cardiac function seen in endotoxin shock and their effect can be diminished by the use of antioxidants (Pattanaik U et al, J “Reactive oxygen species and endotoxic shock: effect of dimethylthiourea”. Cardiovascular Pharmacology Therapy., Vol. 6(3):273-85 Jul. 2001.
Thus, Intravenous NAC becomes an important adjunct in the management of bacterial and viral diseases as well as complications related to such infections such as septic shock, adult respiratory distress and deterioration of cardiac function during endotoxin shock.
The effects of NAC are mediated directly and through the production of increased amounts of intracellular glutathione (Afaq F et al, “N-acetyl L-cysteine attenuates oxidant-mediated toxicity induced by chrysotile fibers,” Toxicology Letters Vol. 117(1-2):53-60, Sep. 30, 2000). Many of the benefits seen in management of viral disease with NAC are mediated through free radical scavenging, particularly of the .OH radical. Removal of this radical performed by glutathione peroxidase, an enzyme that is mediated by the mineral selenium. The importance of selenium in supporting this reaction is demonstrated in a study that demonstrates that a non-lethal virus, Coxsackie virus, becomes lethal in animals deficient in selenium. It was determined that not only was the immune function of the animal altered, the viral genome was altered in such a way that the virus became more pathogenic (Beck M A, “Antioxidants and viral infections: host immune response and viral pathogenicity,” Journal American College Nutrition, 20 (5 Suppl):384S-388S; discussion 396S-397S, October 2001). Selenium 100 mcg to 200 mcg should be given in addition to NAC to maintain and restore Selenium levels. Selenium may administered as organic forms of selenium, such as selenomethionine and selenocysteine, as well as the inorganic forms of the mineral, like sodium selenite and selenate.
Cell studies have demonstrated that a decreased level of glutathione increases sensitivity to radiation (Meister A et al, “Intracellular cysteine and glutathione delivery systems” Journal American College Nutrition 5(2):137-51, 1986). The ability of glutathione and its precursors to moderate the effect of the .OH radical makes NAC an ideal candidate for use in the treatment of radiation exposure. Scavengers of the .OH radical have been demonstrated to protect mammalian animal cells against the damaging effects of radiation (Ewing D et al, “Radiation protection of in vitro mammalian cells: effects of hydroxyl radical scavengers on the slopes and shoulders of survival curves,” Radiation Res, Vol. 126(2):187-97, May 1991). The mechanism is related to the scavenging of the .OH radical as well as other intracellular mechanisms. An evaluation of 35 people with “post radiation syndrome” after exposure to substantial amounts of ionizing radiation (0.01-0.5 Gy) while participating in recovery work in Chernobyl demonstrated that they had a significant decrease in antioxidant defense with a decrease in the activity of glutathione peroxidase and deficiency of selenium (Kumerova A O et al, “Antioxidant defense and trace element imbalance in patients with postradiation syndrome: first report on phase I studies,” Biology Trace Element Research Vol. 77(1):1-12, October 2000).
The “post radiation syndrome” produces headache, dizziness, poor memory, and local pains Id. These symptoms are not necessarily specific of any disease and may be easily confused with the early symptoms of illness such as viral influenza or even viral infection such as smallpox. The early symptoms of anthrax also mimic the early symptoms of viral influenza. Thus, it may be difficult to identify the etiologic cause of the early stage symptoms of biological weapons or radiation. The use of antioxidant support therapy with NAC is an inexpensive way to support an affected individual's antioxidant system while the specific etiology is identified and as an adjunct to antibiotic or antiviral therapy. The NAC antioxidant therapy may be initiated as either oral and/or intravenous therapy. Selenium should also be administered orally at 200 mg per day.
Additional factors also play a role in limiting the individual's ability to combat infection. The stress of infection with either microbial or viral agents will cause a shift in hormone production. This is manifest as a shift in the hypothalamo-pituitary-adrenal axis (HPA) with the resulting production of excess cortisol, the major glucocorticoid. Elevation in cortisol is accompanied by a decrease in cellular immunity in humans undergoing significant stress such as surgery (Tashiro T et al, “Changes in immune function following surgery for esophageal carcinoma,” Nutrition, Vol. 15(10):760-6, October 1999). Stress will also lower the response to vaccination and decrease protection to subsequent infection challenge in pigs immunized with a viral vaccine. Excess of the glucocorticoid is associated with suppression of antiviral immunity (Padgett D A et al, “Steroid hormone regulation of antiviral immunity,” Ann N Y Acad Sci, Vol. 917:935-43, 2000). Counter regulation of immunosuppression due to glucocorticoid elevation would improve immune response and limit virus-mediated damage. Dehydroepiandrosterone (DHEA) and its metabolite Androstenediol (5-androstene-3 beta, 17 beta-diol, AED) have been demonstrated to be protective against lethal infection from Influenza A virus in animals Id. The effect is apparently due to a counterbalance of the immunosuppressive effect of glucorticoids. AED prevents the glucocorticoid-mediated suppression of IL-1, TNF-alpha and IL-2 secretion. An ocular infection model in mice exposed to herpes simplex virus 1 (HSV-1) was favorably modified by the subcutaneous administration of AED (Carr D J, J “Increased levels of IFN-gamma in the trigeminal ganglion correlate with protection against HSV-1-induced encephalitis following subcutaneous administration with androstenediol,” Neuroimmunol Vol. 89(1-2):160-7, Aug. 14, 1998). The dose of AED used in this study was 320 mg/kg. The AED administration results in the increase of chemokines IP-10, MCP-1, the cytokine interleukin-6 (IL-6) and interferon-gamma (IFN-g) and IL-2 and natural killer cell activity, an important component of the removal of cells infected with virus. Thus, DHEA or AED becomes on an important component of antiviral therapy. Samuel et al, U.S. Pat. No. 6,168,804 refer to the use of DHEA as a component of a slow release vehicle to induce TH1 immune response to an immunizing antigen. Daynes et al, U.S. Pat. No. 5,919,465, claim the use of DHEA to augment the immune response due to stress including the stress of viral infection. No patent, however, has claimed the use of a combination of NAC and DHEA or its metabolite AED as an immune enhancing combination for the treatment of viral diseases such as smallpox.
Vaccination after exposure to smallpox has been proposed as part of the management of the disease. Vaccination administered within 4 days of first exposure has been shown to offer some protection against acquiring infection and significant protection against a fatal outcome. (Henderson D A et al, “Consensus statement regarding Smallpox,” Journal American Med Association June 9; 281(22):2127-37, 1999). As the presence of viral infection will decrease the level of glutathione it is likely that the immunization response may be compromised in individuals who are already carrying smallpox. It has been demonstrated that decreased immunologic function in individuals with cysteine or glutathione deficiency can be enhanced by cysteine supplementation (Dröge W et al, “Glutathione and immune function,” Proc Nutrition Soc, Vol. 59(4):595-600, November 2000). Maintaining glutathione levels with glutathione precursors will improve the immune response to vaccination during vaccination for smallpox exposure. It is anticipated that inoculation for smallpox would be under taken at a time of concern about the appearance of smallpox used as a bioterror weapon. In this situation stress will be increased, and the use of the combination of NAC and DHEA is useful to maximize efficient immune response to immunization. The preferred method of use of the invention utilizes the combination of NAC and DHEA prior to or at the time of immunization.